Since the advent of DNA thermal amplification technology, numerous procedures have been developed to amplify polynucleotide sequences on a preparative scale. Of these, the concatamer chain reaction developed independently by Rudert et al. (17a) and White et al. (17b) employ a DNA polymerase-catalyzed thermal amplification system for the generation of DNA concatamers. In this procedure, the primer and template for the amplification reaction are the identical molecule, producing large sequences comprising tandem repeats of the target DNA sequence. Following similar procedures, Louis et al. (17c) prepared isotopically-labeled DNA oligonucleotides for NMR spectroscopy, utilizing labeled deoxynucleotide triphosphates. Although the lafter procedure produced oligonucleotides, amounts of product are still limited and restrict the utility of subsequent studies requiring larger quantities of oligonucleotides. Furthermore, these procedures introduce a degree of heterogeneity in the product, making it unsuitable for certain uses, in particular, high resolution heteronuclear NMR spectroscopy.
Multi-dimensional heteronuclear NMR has become a standard technique to determine the three-dimensional structure of proteins and RNA in solution (1,2). One of the most important advances in the application of NMR spectroscopy to the study of biological systems has been the ease of incorporation of .sup.13 C and/or .sup.15 N into proteins (2-5) and RNA.(6-8). The enrichment of macromolecules in these stable isotopes allows for the dispersion of .sup.1 H, .sup.13 C and .sup.15 N chemical shifts into multiple spectral dimensions in a manner that preserves the chemical and/or spatial relationship between atoms within a molecule of interest (2-5). The resulting enhancement of spectral sensitivity and resolution has had a tremendous impact on the study of chemical and biological phenomena of proteins and RNA. (2-8). In contrast, both the detailed analysis of structure and dynamics of DNA in solution have remained largely inaccessible to the NMR spectroscopist despite the ease of preparation of oligonucleotide duplexes of biological interest. Poor proton density and narrow chemical shift dispersion limits the detailed analysis of structural parameters by homonuclear .sup.1 H-NMR to very small oligonucleotides. While it is desirable to apply heteronuclear NMR to the study of DNA in solution, it has required de novo synthesis of DNA precursors for solid-phase synthesis (9-11). These methods require a certain level of synthetic expertise and are both cost and labor intensive. In this regard, an enzymatic approach would be advantageous and a few such methods have been proposed in recent years (12-17).
It is towards the improvement in the quantity and quality of the large scale preparation of polynucleotide sequences, and in particular, isotopically enriched polynucleotide sequences, and uses thereof, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.